Thanks again!
]]>I didn't have a positive control in the last transformations I tried for RF but I used the cells from the same batch for a regular Maxi prep and they worked fine and the transformation protocol hasn't changed. I should probably include a control in subsequent transformations just to rule it out.
I used the entire PCR reaction to transform. Was that correct? I assumed that as it is a rare event it would be better to use all of it...
There was absolutely nothing on either plates and I know I've got the right antibiotic.
]]>I'm guessing you had a positive control that came up as expected? And did you use the entire PCR reaction in the transformation, or just 1 μl (i.e., 1 μl good; 20 μl bad)? I'm surprised you didn't get any negative colonies at all using the OneShot Max cells, because they are crazy sensitive.
]]>I transformed the DpnI digested PCR into OneShot MAX efficiency DH5a chemically competent cells from Life Tech. using the standard 42 degrees heat shock protocol. Shook them for half an hour in SOC at 37 then plated the whole volume onto two LB+Amp plates
]]>ps. I've linked the project I think was yours to your account. Go into the project management dashboard and see if it's right.
]]>I really like the RF cloning protocol and have been trying to swap the V5/His tag in the pEF1 vector with an mNeptune sequence. My first PCR works really well and I get good yield from the gel extraction but when I try the 2nd PCR I get no colonies at the end. The 2nd PCR product is treated with DpnI which must work as I don't get any false positives. I have tried both the 2-step and the 3-step with 60 degrees annealing temperature but to no avail. The only thing that I can think of is something to do with the plasmid annealing part of the megaprimer. I'm not sure whether the details provided by the website are on record somewhere...
I hope you can help.
BW,
Zac